Mast cells (MCs), granulocytes, and lymphocytes are integral to the development of an allergic response. Allergic inflammation may also be generated through activation of receptors coupled to heterotrimeric G proteins (GPCRs). The purpose of this study is to understand mechanisms of G protein-mediated signal transduction in immune cells, with a focus on GPCR-mediated trafficking of leukocytes to sites of allergic inflammation. GPCRs activate a core pathway of heterotrimeric G proteins, which bind guanosine triphosphate (GTP) in exchange for guanosine diphosphate (GDP). The GTP-bound form of the G protein alpha subunit induces downstream signaling cascades, including intracellular calcium flux responsible for MC/basophil degranulation. This project focuses on a family of regulators of G protein signaling (RGS proteins), which inhibit the function of G alpha-i and G alpha-q, but not G alpha-s, proteins by increasing their GTPase activity. G alpha subunits oscillate between GDP- (inactive) and GTP- (active) bound forms based on ligand occupancy of the associated receptor. The GTPase accelerating (GAP) activity of RGS proteins limits the time of interaction of active G-alpha and its effectors, resulting in desensitization of GCPR signaling. Despite a growing body of knowledge concerning the biochemical mechanisms of RGS action, relatively little is known about the physiological role of these proteins in allergic inflammation. A major area of investigation is the recruitment of inflammatory cells to sites of inflammation. Chemokines are a major class of compounds acting on leukocyte GPCRs, which orchestrate immune cell trafficking, and RGS proteins including RGS5, RGS13, and RGS16 inhibit chemokine signaling by desensitizing GPCR signals. A second research area is the trafficking of mast cells and granulocytes during allergic responses. Many allergens contain intrinsic proteolytic activity and bind protease activated GPCRs. Although sensitization to protease allergens, such as papain, helminth infection, chronic allergic skin inflammation, and nasal rhinitis are associated with basophil recruitment to inflamed tissue or to draining lymph nodes (LNs), the precise role of basophils and mechanisms involved in their recruitment is incompletely understood. We are generating mouse strains containing mast cells or basophils hyper- or hyporesponsive to chemokines in order to study the contribution of these cells to various allergic responses. In FY16, we contributed key experiments to a collaborative study with Dr. Rosenberg (LAD/IIS) to characterize chemoattractant properties of an RNAse family member (mEar 11), which is expressed in macrophages and is upregulated by Th2 cytokines (IL-4/13). We demonstrated that mEar11 acts as a potent chemoattractant for macrophages. This property did not depend on mEar11 RNase activity. Further investigation of mEar11 in models of allergic disease may shed light on the function of these leukocytes at sites of tissue inflammation. In another collaborative project with Dr. Khasawneh (Western University), we demonstrated a critical negative regulatory function of RGS16 in platelet activation. Platelets from RGS16-deficient mice exhibited enhanced aggregration, granule secretion, and adhesion in response to GPCR ligands thrombin and CXCL12 as well as collagen. Patients with undefined immunodeficiencies and novel mutations in G proteins and/or RGS proteins are being characterized in collaborative studies with Drs. Orange and Su. A final area of investigation is the role of RGS5 in neutrophil trafficking. Using Rgs5-/- mice, we discovered in 2016 that neutrophils deficient in RGS5 do not traffic normally to sites of inflammation. Neutrophils isolated from Rgs5 gene deleted mice display enhanced chemotaxis to proinflammatory chemokines. Current studies are aimed at understanding the molecular mechanisms underlying this phenotype and determining whether the defects are leukocyte-intrinsic.